EP1399556A2 - Epitopes antigenes du factor viii, inhibiteurs diriges contre ces epitopes et leur utilisation - Google Patents

Epitopes antigenes du factor viii, inhibiteurs diriges contre ces epitopes et leur utilisation

Info

Publication number
EP1399556A2
EP1399556A2 EP02732243A EP02732243A EP1399556A2 EP 1399556 A2 EP1399556 A2 EP 1399556A2 EP 02732243 A EP02732243 A EP 02732243A EP 02732243 A EP02732243 A EP 02732243A EP 1399556 A2 EP1399556 A2 EP 1399556A2
Authority
EP
European Patent Office
Prior art keywords
ser
asp
epitope
thr
arg
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP02732243A
Other languages
German (de)
English (en)
Other versions
EP1399556B1 (fr
Inventor
Ruth Laub
Mario Di Giambattista
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CAF - DCF CVBA - SCRL
Original Assignee
Departement Central de Fractionnement de la Croix-Rouge SCRL
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Departement Central de Fractionnement de la Croix-Rouge SCRL filed Critical Departement Central de Fractionnement de la Croix-Rouge SCRL
Publication of EP1399556A2 publication Critical patent/EP1399556A2/fr
Application granted granted Critical
Publication of EP1399556B1 publication Critical patent/EP1399556B1/fr
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in eptitope analysis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/745—Assays involving non-enzymic blood coagulation factors
    • G01N2333/755—Factors VIII, e.g. factor VIII C [AHF], factor VIII Ag [VWF]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/806—Antigenic peptides or proteins

Definitions

  • the present invention relates to the antigenic polypeptide sequences (epitopes) of factor VIII to the anti-FVIII inhibitors which are directed against these sequences and to anti-inhibitors which are directed against said anti-FVIII inhibitors.
  • the present invention also relates to a pharmaceutical composition and to a diagnostic device comprising at least one of the above mentioned molecules.
  • FVIII is a large multi-domain protein of 2,332 amino acids made up of three structural domains, A, B and C which are arranged in the order Al:al:A2 :a2 :B:a3 :A3 :C1:C2.
  • the A domains possess more than 40% homology and are also homologous to ceruloplasmin (for recent review, see Pratt (2000) and Saenko (1999)). 30% homology also exists between the A domains of factor V and FVIII.
  • the C domain occurs twice and is reported to be able to bind glyco-conjugates and phospholipids having a net negative charge. It exhibits homology with lectins which are able to bind to negatively charged phospholipids .
  • the platelet attachment site has been located in this region (C2 domain) (Foster et al . , (1990)).
  • These antigenic determinants consist of fragments 351 - 365 (Al domain - heavy chain) , 713 - 740 (A2 domain) , 1670 - 1684 (A3 domain - light chain) (NH 2 end of the light chain) or else 2303 - 2332 (C2 domain - light chain) (Foster C, (1990)), fragments 701 - 750, 1663 - 1689, 330 - 472, 1694 - 1782 (EP-0 202 853), 322 - 740 and 2170 - 2322.
  • the U.S patent 5,744,446 describes an hybrid human/animal Factor VIII having a sequence of amino acids selected from the group of the A2 domain fragments 373-540, 373-508, 445-508, 484-508, 404-508, 489-508 and 484-489, with corresponding sequences of porcine or urine Factor VIII, said hybrid being used for the treatment of Factor VIII deficiencies.
  • the antibodies which recognize these various sites interfere, with the activation of FVIII, the binding of v f, FIXa, FXa, APC or phospholipids.
  • the specific antibody response to FVIII vary considerably among individuals, and epitopes for inhibitor antibodies have to be determined for all FVIII domains (see for recent review Scandella, 2000; Lollar, 2000).
  • anti-FVIII autoantibodies are rare in non-hemophilic patients (annual frequency in the population: 1-5/10 6 ) (Morrisson and Ludlam) (1995) . They are associated with a number of autoimmune diseases and are often characterized by life-threatening hemorrhage. On the other hand, anti-FVIII antibodies have also been described in healthy subjects (Algiman et al, 1992; Moreau et al, 2000) , without any apparent effect on the subjects' levels of circulating FVIII.
  • Self proteins or derived peptides may elicit an immune response if presented to CD4 T cells at inflammatory sites by professional antigen presenting cells.
  • FVIII domains were recognized: A3 domain was recognized more strongly and frequently and each domain forms several epitopes .
  • FVIII FVIII
  • PCC activated PCC
  • FVIIa FVIII inhibitor activity
  • porcine FVIII may be used to achieve haemostasis in patients with antibodies that do not substantially crossreact with porcine FVIII before or during the treatment (Lollar, 2000) .
  • a potential alternative approach to inhibit the production of inhibitors is blockade of the T cell/B cell collaboration mediated by through receptor ligand binding signal events (Ewenstein et al, 2000) . Preliminary clinical trials were performed using a humanized mouse monoclonal antibody to human T cell CD40 ligand (CD 154) . [0016] A profitable strategy for reducing the level of inhibitors has consisted in subjecting patients to an extracorporeal circulation to enable solid-phase absorption of the total IgG.
  • the immunoabsorbant could be sepharose-bound staphylococcal protein A or sepharose-bound polyclonal sheep antibodies to total human immunoglobulin (Knobf and Derfler, 1999) .
  • the foreign proteins protein A, sheep anti-human Ig
  • ICH Topic Q5A, Directive 92/79/EC ICH Topic Q5A, Directive 92/79/EC
  • IVIG polyvalent intravenous immunoglobulins
  • the present invention aims to obtain new antigenic epitopes of factor VIII in order to improve the diagnosis and/or the therapy (including the prevention) of immune disorders (in particular those induced by inhibitors of FVIII, especially inhibitors of the binding of FVIII to the von Willebrand factor (v f) , to the FIX and/or to membrane phospholipids (PL) ) , said epitopes allowing a screening between non-inhibitory and inhibitory anti-FVIII allo- or auto-antibodies (allo- or auto-immunoglobulins) .
  • Another aim of the invention is to obtain inhibitors which exhibit an immunoaffinity with these antigenic epitopes, as well as to obtain anti-inhibitors, in particular antibodies or (T) cell receptors, which are directed against the abovementioned said inhibitors and whose purpose is to improve the diagnosis and/or therapy (or prevention) of immune disorders .
  • a further aim of the invention is to obtain said molecules at high purity, in industrial level, without contaminants (viruses, prions,...) and according to the GMP practices in the field of therapy and diagnostics (ICH topic QSA, Directive 92/79/EC, etc.).
  • the present invention relates to the antigenic polypeptide sequences (epitopes) of factor VIII whose complete sequence is described by Verhar et al . (1984) and which provides the reference for the amino-acids numerotation of the complete factor VIII sequence.
  • the "complete polypeptide sequence of factor VIII" is understood to be the natural human or animal sequence, which may be glycosylated and which has been obtained by purification from pools of plasma, in particular cryoprecipitate, by synthesis and/or by genetic manipulation (sequence from which portions which are not involved in the mechanism of blood coagulation may have been deleted) of factor VIII.
  • the present invention relates, in particular, to an antigenic epitope sequence of factor VIII which is selected from the group consisting of : the epitope comprised between serine 2018 and histidine 2031 inclusive, defined by the following sequence: SEQ ID No: 10:
  • Ser Asp Asp Pro Ser Gly Ala lie Asp Ser Asn Asn Ser 1 5 10 the epitope asparagine .2128 to asparagine 2138 inclusive , defined by the following sequence :
  • SEQ ID No: 32 Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr 1 5 10 possibly deleted from one or more amino acids of the terminal tripeptide Thr-Arg-Tyr involved in the phospholipid von Willebrand factor binding site - the epitope glutamic acid 2322 to tyrosine 2332 inclusive, defined by the following sequence (P16) : SEQ ID No: 33 :
  • the invention also relates to the major parts of the said epitopes .
  • Said epitopes can be deleted from one or more terminal amino acids, preferably from one, two or three amino acids, or can be replaced by one or more amino acids that present the same characteristic of hydrophilicity, flexibility and accessibility.
  • epitopes according to the invention are comprised in major determinants of human inhibitors epitopes or several factors binding sites or binding sites of known monoclonal antibodies, especially the portion C2 that is known to be the binding site of the monoclonal antibody Mas531P or the binding site ESH8 as .well as phospholipids, Factor Xa or the von Willebrand factor binding site.
  • the specific epitopes according to the invention or their major parts are preferred selected portions of said binding sites or may include a possible overlapping with said binding sites .
  • Another aspect of the present invention is related to a modified (recombinant or transgenic) FVIII, possibly obtained by genetic engineering, and deleted from one or more of the above-identified epitopes or major parts of said epitopes.
  • said FVIII still allows the binding of coagulation factor (s), but will be less immunogenic and will not induce or induce less the formation of inhibitors directed against said modified FVIII or natural FVIII.
  • said epitopes are also independently immunogenic (that is to say they are immunogenic even without being complexed with a protein of large size such as BSA, KLH, haemocyanin, etc.), and preferably exhibit an immunoaffinity within inhibitors of factor VIII, such as anti-factor VIII antibodies, and/or exhibit an immunoaffinity for the receptors of the T lymphocytes and possibly B lymphocytes.
  • a protein of large size such as BSA, KLH, haemocyanin, etc.
  • epitopes and/or major parts of said epitopes induce an immune reaction (antibody synthesis) when they are injected into a rabbit.
  • Said sequences are unexpectedly characterized by substantial immunogenicity towards monoclonal and polyclonal antibodies, but are sufficiently short to be readily and advantageously obtained by synthesis .
  • the present invention also relates to the conformational epitopes which comprise at least two different sequence epitopes and/or at least two major parts of said epitopes according to the invention and above identified.
  • the conformational epitopes are made up of two or more different portions of a polypeptide sequence, which portions are located in proximity to each other when the protein is folded in its tertiary or quaternary structure .
  • These epitopes are capable of being “recognized” (that is to say of exhibiting an immunoaffinity) , preferably simultaneously, with inhibitors of factor VIII, in particular B and T lymphocytes (by way of the major histocompatibility locus (MHC I and/or II) ) and/or anti-factor VIII antibodies (Scandella et al . (2000); Reding et al . (2000)).
  • the said epitopes and/or the major parts of said epitopes are complexed with a carrier protein or a carrier peptide, such as BSA, or KLH haemocyanin, as to form a complex exhibiting a more powerful immunogenicity.
  • a carrier protein or a carrier peptide such as BSA, or KLH haemocyanin
  • the present invention is also related to a pool of antigenic epitopes of factor VIII which comprises a mixture of the epitopes above mentioned linear epitopes
  • SEQ ID 10 SEQ ID 17, SEQ ID 20, SEQ ID 21, SEQ ID 24, SEQ ID 27, SEQ ID 30, SEQ ID 31, SEQ ID 32, SEQ ID 33
  • conformational epitopes made of said epitopes or a pool which may comprise at least one of said epitopes and one or more additional antigenic epitopes of factor VIII already described in the state of the art and preferably selecting from the group consisting of : the epitope arginine 1648 to tyrosine 1664 inclusive, defined by the following sequence: SEQ ID No:l: Arg Asp He Thr Arg Thr Thr Leu Gin Ser Asp Gin Glu Glu He Asp
  • Glu Asp Asp Lys Val 20 possibly deleted from the terminal amino acids alanine and valine (PI) the epitope glutamic acid 181 to leucine 192 inclusive , def ined by the following sequence : SEQ ID No : 12 :
  • Asp Arg Asp Ala Ala Ser Ala Arg Ala 20 25 possibly deleted from one or more amino acids of the nonapeptide Asp-Arg-Asp-Ala-Ala-Ser-Ala-Arg-Ala the epitope aspartic acid 327 to methionine 355 inclusive , defined by the following sequence : SEQ ID No : 14 : Asp Ser Cys Pro Glu Glu Pro Gin Leu Arg Met Lys Asn Asn Glu Glu
  • He Gly Arg Lys Tyr Lys Lys 20 possibly deleted from one or more amino acids of the tetrapeptide Asp -Asp -Ar -Ser (P3 ) , the epitope valine 517 to arginine 527 inclusive, defined by the following sequence:
  • epitope isoleucine 2081 to serine 2095 inclusive defined by the following sequence :
  • Another aspect of the present invention relates to an inhibitor of factor VIII which exhibits an immunoaffinity with antigenic epitopes, with the major parts of said epitopes and/or with the complex according to the invention.
  • An inhibitor is understood to mean any biological molecule or cell (such as a T-lymphocyte) binding to said FVIII and capable of giving rise to immune disorders (characterized by humoral immune response and/or cellular immune response against said FVIII) .
  • an inhibitor can be an anti-factor VIII monoclonal or polyclonal antibody or antibody fragment (such as the hypervariable Fab portion of the said antibody) which inactivates the said factor VIII and/or which inhibits the binding of factor VIII to the von
  • Willebrand factor and/or to membrane phospholipids Willebrand factor and/or to membrane phospholipids.
  • the said inhibitors are synthesized by a "chimaeric" animal which comprises a human immune system, such as an hu-SCID mouse or transgenic mouse producing human antibodies or other antibodies production technologies as phage display technology or immortalized B- cells, by EPV in particular.
  • a human immune system such as an hu-SCID mouse or transgenic mouse producing human antibodies or other antibodies production technologies as phage display technology or immortalized B- cells, by EPV in particular.
  • Another aspect of the invention relates to an anti-inhibitor which is directed against the said previously described factor VIII inhibitor.
  • an anti-inhibitor which is directed against the factor VIII inhibitor is understood to mean any chemical or biological molecule, a cell and/or a cell fragment (receptor) which is capable of interfering with the said inhibitor in such a way as to ensure its inactivation or avoid or reduce its binding to the factor VIII.
  • an anti-inhibitor is an anti-anti-factor VIII idiotype (monoclonal or polyclonal) antibody or antibody fragment, natural or obtained by genetic engineering .
  • Another aspect of the invention relates to a pharmaceutical composition which comprises an adequate pharmaceutical carrier or a diluant and an element selected from the group consisting of said epitopes or a pool thereof, an inhibitor of factor VIII which is directed against them, an anti-inhibitor which is directed against the said inhibitor, and/or a mixture of these.
  • the type and amount of adequate pharmaceutical carrier or diluant (and possibly adjuvant or excipient) present in said pharmaceutical composition may vary according to the method of administration and is possibly combined an adjuvant in order to improve therapeutical properties of the pharmaceutical composition according to the invention or to reduce its possible side effects.
  • Suitable pharmaceutical acceptable carriers used in the pharmaceutical composition according to the invention are well known by the person skilled in the art and are selected according to the methods generally applied by pharmacists and may include solid, liquid or gaseous non-toxic pharmaceutically acceptable carriers.
  • the percentage of active product / pharmaceutical acceptable carrier may vary within very large ranges only limited by the tolerance and the possible side effects on ' patients (including humans) , and by frequency and/or mode of administration.
  • a diagnostic and/or purification device such as a diagnostic kit, an affinity filter, or a chromatography column which comprises an element which is selected from the group consisting of these epitopes and/or major parts of said epitopes, the complex according to the invention or a pool thereof, an inhibitor which is directed against them, an anti-inhibitor which is directed against said inhibitor, and/or a mixture of these.
  • said device comprises the pool of said epitopes which allow a screening of patients and may detect the most important inhibitors present in said patients and which allow a positive test with enough specificity and sensibility.
  • the purification device can therefore consist of a chromatography column which comprises these epitopes and/or major parts of epitopes, attached to the solid phase of the chromatography column.
  • a physiological liquid such as serum
  • which is derived from a patient and which comprises inhibitors of factor VIII pass through a solid support (chromatography column) , with said inhibitors (for example antibodies) becoming attached specifically to said epitopes or said major parts or a pool thereof.
  • said inhibitors for example antibodies
  • anti-inhibitors anti-anti-factor VIII idiotype antibodies
  • the present invention is also related to a method of treatment (ex vivo treatment) of a patient suffering from a pathology induced by inhibitors to the factor VIII which comprises the steps of extracting said physiological liquid (blood or serum) from the patient, obtaining its reaction upon a solid support binding the epitopes or a pool thereof according to the invention and reinjecting said physiological liquid to the patient after the removing of the inhibitors having fixed said epitopes, majors parts or a pool thereof.
  • a final aspect of the invention relates to the use of the pharmaceutical composition according to the invention for preparing a medicament used for preventing and/or treating immune disorders, in particular those induced by inhibitors of factor VIII, inhibitors of the binding of factor VIII to the factor IX and/or the factor X and/or the von Willebrand factor (vWF) and/or inhibitors of the binding of factor VIII to membrane phospholipids.
  • immune disorders in particular those induced by inhibitors of factor VIII, inhibitors of the binding of factor VIII to the factor IX and/or the factor X and/or the von Willebrand factor (vWF) and/or inhibitors of the binding of factor VIII to membrane phospholipids.
  • vWF von Willebrand factor
  • Figure 1 depicts the hydrophilicity, flexibility and accessibility graph of the A3 sequence of Factor VIII renumbered 1 to 371 amino acids (surface value for each amino acid) .
  • Figure 2a represents the elution ' profile related to the purification of human anti-SEQ ID 32 antibodies by affinity chromatography on peptide-Sepharose column.
  • Cohn fraction II+III solution 50 ml was loaded onto the column (1 ml gel) at a flow rate of 1 ml/min. The separation of specific antibodies was performed as described hereafter.
  • the arrow indicates the position of specific human anti-SEQ ID 32 antibodies.
  • Figure 2b represents FVIII clotting activity in the presence of anti- (SEQ ID 32) IgG purified from Cohn fraction II+III. The clotting activity of FVIII was measured as described hereafter in the presence of increasing amount of anti-SEQ ID 32.
  • the % of FVIII activity (FVIII activity in the presence of antibody/FVIII activity in absence of antibody) *100.
  • Figure 3 represents the human anti-peptide antibody immunoreactions with FVIII polypeptides after western blotting (panel A from left to right : human antibodies HAP1 through HAP4, specific for different FVIII epitope sequences found in the FVIII HC - see also table 2 and panel B : human antibodies specific for the P5 peptide and the FVIII LC sequences, P7, P8 and P9 - see also table 2) .
  • the RAP9 lane shows the reactivity of FVIII polypeptides towards purified rabbit antibodies specific for the peptide sequence Arg 1797 -Tyr 1815 (see also table 2) .
  • Figure 4 represents ELISA reactivity of 4 inhibitor plasmas with different peptide sequences. Inhibitors present in 4 patients plasmas were analyzed by ELISA test using as coated antigens the different selected FVIII epitopes synthetic peptides as indicated in ordinate .
  • MAS530p (Harlan-Seralab, Indianapolis, IN) is a mouse monoclonal antibody specific for the 44-kDa A2 domain of the factor VIII heavy chain.
  • Biotin-labeled rabbit IgG anti-mouse IgG was purchased from Dakopatts
  • IgG and biotin-labeled mouse IgG anti-rabbit IgG were obtained from Sigma Chemicals (St Louis, MI) , purified ⁇ - thrombin (3000 IU/mg) , streptavidin-peroxidase conjugate, ovalbumin (OVA) , bovine serum albumin (BSA) , keyhole limpet haemocyanin (KLH) , and o-phenylenediamine (OPD) were purchased from Sigma Chemicals (St. Louis, MI). Casein was obtained from Merck (Darmstadt, Germany) . 4-chloro- 1- naphtol and biotinylated molecular weight markers were obtained from Bio-Rad Laboratories (Hercules, CA) . Freund's adjuvant was from Difco (Detroit, Michigan) .
  • Plasma FVIII was a solvent/detergent-treated FVIII concentrate (100 IU/mg protein) purified by ion exchange chromatography (FVIII Cone. SD, CAF-DCF- Red Cross , Brussels, Belgium) .
  • Albumin- free recombinant FVIII was obtained from Hyland (Glendale, CA) .
  • Plasma fraction immunoglobulins were obtained from Hyland (Glendale, CA) .
  • Cohn Fraction II+III was obtained from large plasma pool from 4,800 unpaid donors, after precipitation in the presence of increasing ethanol concentration. This fraction contains all Ig classes and subclasses. IgG composition was determined by nephelometry. The 'relative percentage of each subclass was 63,7; 30,1; 3,4 and 2,8 for IgGl, IgG2, IgG3 and IgG4 respectively (average values for 3 different batches of FII+III) .
  • Factor VJJJ concentrates/ Factor VIII activity and activity- inhibition
  • Factor VIII activity was determined in a one- stage clotting assay adapted for use on the Coagulometer KC4A (Sigma Diagnostics) .
  • the assay uses severe hemophilia A plasma (Organon Teknika, Cambridge, UK) and APPT reagent from Instrumentation Laboratory (Warrington, UK) . Potencies were calculated relative to the 5 th International Standard FVIII concentrate 88/640 (5.4 IU/ l) (NIBSC, Potters Bar, UK).
  • FVIII- inhibitory activity was measured in purified rabbit and human IgG preparations according to the modified Bethesda assay. Briefly, affinity-purified IgGs were serially diluted and incubated for 1 h in the presence of FVIII concentrate 88/640 (1 IU/ml) at 37°C. The residual FVIII activity was measured as described above.
  • samples were dialyzed 3 times against 5 volumes of TE buffer (20 mM Tris-HCI pH 7.2, 150 mM NaCl and 0.02% NaN 3 ) and loaded onto the column at a flow rate of 1 ml/min.
  • the column was sequentially washed at 2 ml/min with 50 ml TE buffer and 30 ml TE containing 1 M NaCl.
  • the material was eluted (1 ml/min) with 5 ml of 0.1 M citric acid pH 2.5 and directly recovered in 5 ml of 1M Tris-HCl, pH 9 . 0 .
  • Samples were finally dialyzed versus 10 volumes of equilibration buffer and concentrated on Centriprep-30 (Amicon, Beverly, MA) . Ig recovery was determined by the Bio-Rad protein assay.
  • Results are summarized in Table 1 which concerns the characterization of rabbit anti-FVIII-peptide antisera and recovered affinity-purified of immunoglobulins.
  • the FVIII domain recognized by the anti-FVIII peptide Ig after immunoblotting is shown in (column d, Table 1) and Ig protein recoveries (column e, Table 1) were measured using immunoglobulins as the standard.
  • the inhibitory activity, expressed in BU/mg protein, was determined in a FVIII neutralizing activity assay (column f , Table 1) .
  • FVIII anti-peptide antisera The reactivity of FVIII anti-peptide antisera was measured by an ELISA using, as antigen, either the different corresponding FVIII -peptide coupled to KLH protein or purified rFVIIl.
  • the binding reaction of each anti-FVIII -peptide antiserum was specific both for the FVIII peptide used to elicit the immune response in rabbit and for rFVIIl (see Table 1) .
  • the specific rabbit IgG were purified by affinity chromatography on peptide-Sepharose as described under Methods. When FVIII -neutralizing activity was measured in a one-stage clotting assay, significant inhibition was found with two rabbit IgG purified preparations: RAP2 , corresponding to IgG specific for SEQ ID No. 14 and RAP7 specific for SEQ ID No: 01.
  • rFVIIl and the rFVIIl fragments obtained after treatment with thrombin were resolved by SDS-PAGE and analyzed by western blotting with different preparations of rabbit IgGs (RAPl to RAP17 Igs) .
  • Antisera RAPl, RAP2 , RAP3 , RAP4 recognized exclusively the heavy chains (200 kDa to 92 kDa) . Antisera RAPl and RAP2 reacted with the 50-kDa Al- domain fragment; RAP3 and RAP4 bound to the 44-kDa fragment
  • domain A2 domain A2
  • RAP5 specific for the B domain bound to the high-molecular-weight FVIII heavy chain (about 200 -kDa) .
  • RAP7, RAP8, and RAP9 reacted with the 80 -kDa light-chain doublet.
  • RAP9 and RAP12 to RAP17 antibodies also detected the 72 -kDa FVIII light-chain fragment.
  • each reactive antiserum showed a strong reaction with the corresponding FVIII fragment containing the selected linear epitope. No reaction was detectable in the gels between RAP6 or RAP10 and the HC or LC FVIII fragments.
  • Table 2 concerns the characterization of human anti-FVIII antibodies from Cohn fraction II+III of healthy individuals.
  • Binding to the rFVIIl HC or LC chains and to the rFVIIl thrombin fragment is shown respectively in columns c and d,
  • FVIII FVIII, recombinant FVIII, and the fragments obtained after thrombin proteolysis .
  • FVIII fragments were identified with either FVIII-HC- or FVIII-LC-specific mouse monoclonal antibodies or FVIII-peptide-specific rabbit polyclonal antibodies.
  • the human antibodies were identified after binding of biotinylated goat anti-human IgG.
  • Figure 3 shows the immunoreaction of high-molecular-weight FVIII
  • Lys 425 or SEQ ID No : 19 (Cys 711 -Asp 725 ) .
  • the 50-kDa FVIII fragment (domain Al) was recognized by human antibodies purified on Ser 109 - Lys 127 or Cys 329 -Asp 348 -Sepharose and the 44-kDa FVIII fragment (A2) by immunoglobulins purified on Tyr 07 -Lys 425 and Cys 711 -Asp 725 -Sepharose .
  • Epitope Arg 1652 -Tyr 1654 and the adjacent major binding site vWF are located in the acidic light-chain peptide a.3. As shown by western blotting, a.3 is released from the A3 domain after thrombin treatment, preventing further binding of anti- (Arg 1552 - Tyr 1664 ) Ig to activated FVIII. Similar results have been reported by Shima et al (1991) , who described the FVIII sequence Asp 1563 -Ser 1669 as a binding site of rabbit polyclonal antibodies neutralizing FVIII activity.
  • Epitope Cys 329 -Asp 348 overlapped the acidic Asp 3 8 -Lys 3 ⁇ 2 sequence (in al) described as adjacent to the activated protein C (Arg 336 ) and thrombin (Arg 372 ) cleavage sites. It is the target of human hemophilic inhibitors.
  • Anti- (Asp 348 -Lys 362 ) antibodies may interfere with proteolysis or with the FX interaction site (Met 337 -Arg 372 ) (Saenko et al . , 1999 and Scandella et al . , 2000).
  • FVIII -neutralizing activity was measured in all 13 Ig preparations. Seven human Ig preparations displayed inhibition of procoagulant activity, these being specific for amino-acid residues Cys 711 -Asp 725 , Tyr 1681 - Arg 1696 , and Arg 1797 -Tyr 1815 respectively.
  • the Cys 711 -Asp 725 sequence contains sulfated tyrosines at Tyr 718 , Tyr 719 , and Tyr 723 , and overlaps with the FVIII HC region Lys 713 -Arg 740 described as promoting both activation and HC proteolysis.
  • the additional sulfated groups may be required for proper interaction with thrombin or another component as in the FX-activating complex.
  • Peptide P8 (Tyr 1681 -Arg 1S95 ) (FVIII LC) includes the sequence Glu 1684 -Arg 1689 already described by Shima et al, 1991. It contains the thrombin activation site Arg 1689 -Ser 1690 .
  • p4 (Cys 711 -Asp 725 ) is also included in the Asp 712 -Ala 736 sequence detected by analysis of the patient antibody repertoire by gene phage display technology. It is proposed as a possible additional inhibitor in patients (van den Brink et al, 2000) .
  • Peptide P9 (Arg 1797 -Tyr 1815 ) contains the FXa binding site (see below) .
  • FVIII epitope sequences help to determine the contribution of patient polyclonal anti-FVIII Igs to overall inhibitory and regulatory activity. They could also be used to monitor the usual switch in anti-FVIII specificity in a patient during treatment . Said characterization of FVIII epitopes and a model of their locations on the folded molecule improves the treatment of inhibitors in both hemophilic and non- hemophilic patients (detection, follow-up, therapeutic use of FVIII epitope peptides%) .

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Urology & Nephrology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Food Science & Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Rheumatology (AREA)
  • Mycology (AREA)
  • Diabetes (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne une séquence de polypeptides antigènes (épitopes du factor VIII), des inhibiteurs dirigés contre cette séquence, et des anti-inhibiteurs dirigés contre ces inhibiteurs. L'invention concerne également une composition pharmaceutique et un dispositif de diagnostic comprenant l'une au moins de ces molécules.
EP02732243A 2001-05-09 2002-05-06 Epitopes antigenes du factor viii, inhibiteurs diriges contre ces epitopes et leur utilisation Expired - Lifetime EP1399556B1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US853080 2001-05-09
US09/853,080 US20020068303A1 (en) 1994-07-14 2001-05-09 Antigenic polypeptide sequences of factor VIII, and fragments and/or epitopes of these sequences
PCT/BE2002/000070 WO2002090542A2 (fr) 2001-05-09 2002-05-06 Epitopes antigenes du factor viii, inhibiteurs diriges contre ces epitopes et leur utilisation

Publications (2)

Publication Number Publication Date
EP1399556A2 true EP1399556A2 (fr) 2004-03-24
EP1399556B1 EP1399556B1 (fr) 2011-09-21

Family

ID=25314986

Family Applications (1)

Application Number Title Priority Date Filing Date
EP02732243A Expired - Lifetime EP1399556B1 (fr) 2001-05-09 2002-05-06 Epitopes antigenes du factor viii, inhibiteurs diriges contre ces epitopes et leur utilisation

Country Status (7)

Country Link
US (2) US20020068303A1 (fr)
EP (1) EP1399556B1 (fr)
JP (1) JP4500494B2 (fr)
AT (1) ATE525470T1 (fr)
CA (1) CA2446390C (fr)
ES (1) ES2372811T3 (fr)
WO (1) WO2002090542A2 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2482926A1 (fr) * 2002-04-18 2003-10-23 Merck Patent Gesellschaft Mit Beschraenkter Haftung Facteur viii modifie
GB0713187D0 (en) * 2007-07-06 2007-08-15 Cambridge Entpr biomolecule binding ligands
GB0801513D0 (en) * 2008-01-28 2008-03-05 Circassia Ltd Peptides from factor VIII

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4649132A (en) 1983-03-31 1987-03-10 Scripps Clinic And Research Foundation Treatment of Factor VIII inhibitors
US4965199A (en) * 1984-04-20 1990-10-23 Genentech, Inc. Preparation of functional human factor VIII in mammalian cells using methotrexate based selection
KR910006424B1 (ko) 1985-08-21 1991-08-24 인코텍스 비.브이 편성브리프(brief) 제조방법
BE1008491A3 (fr) 1994-07-14 1996-05-07 Croix Rouge De Belgique Depart Sequence polypeptidique antigenique du facteur viii, fragments et/ou epitopes de celle-ci.
US6500646B1 (en) * 1996-12-27 2002-12-31 Mochida Pharmaceutical Co., Ltd. Cell membrane-directed drugs
EP1051432B1 (fr) * 1998-12-08 2007-01-24 Biovation Limited Modification de l'immunogenicite de proteines
US20040096456A1 (en) * 2000-12-01 2004-05-20 Conti-Fine Bianca M Method to treat hemophilia

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO02090542A2 *

Also Published As

Publication number Publication date
WO2002090542A2 (fr) 2002-11-14
EP1399556B1 (fr) 2011-09-21
US20020068303A1 (en) 2002-06-06
CA2446390A1 (fr) 2002-11-14
US7981865B2 (en) 2011-07-19
CA2446390C (fr) 2012-01-17
JP2004535393A (ja) 2004-11-25
ATE525470T1 (de) 2011-10-15
JP4500494B2 (ja) 2010-07-14
ES2372811T3 (es) 2012-01-26
WO2002090542A3 (fr) 2003-12-24
US20060051367A1 (en) 2006-03-09
WO2002090542A8 (fr) 2004-02-12

Similar Documents

Publication Publication Date Title
Gilles et al. Anti-factor VIII antibodies of hemophiliac patients are frequently directed towards nonfunctional determinants and do not exhibit isotypic restriction
Gilles et al. Healthy subjects produce both anti-factor VIII and specific anti-idiotypic antibodies.
Gilles et al. Neutralizing antiidiotypic antibodies to factor VIII inhibitors after desensitization in patients with hemophilia A.
AU626255B2 (en) Factor viii binding domain of von willebrand factor
Abumiya et al. An anti-tissue factor pathway inhibitor (TFPI) monoclonal antibody recognized the third Kunitz domain (K3) of free-form TFPI but not lipoprotein-associated forms in plasma
Villard et al. Peptide decoys selected by phage display block in vitro and in vivo activity of a human anti-FVIII inhibitor
JP2009528285A (ja) ファクターｖｉｉｉのｃ１ドメインに対する阻害抗体の阻害活性を中性化する抗イデオタイプ抗体
JPH0638790A (ja) ファクターｖｉｉｉ：ｃ凝固因子ポリペプチド類に特異的なモノクローナル抗体
JP2006316062A (ja) 因子ｖｉｉｉの抗原ポリペプチド配列、その断片および／またはエピトープ
ES2377128T3 (es) Anticuerpo antiidiotípico que neutraliza la actividad inhibidora de un anticuerpo inhibidor del factor VIII
Gilles et al. Some factor VIII (FVIII) inhibitors recognise a FVIII epitope (s) that is present only on FVIII-vWF complexes
US7981865B2 (en) Antigenic fragments of human factor VIII polypeptides
Mühle et al. Epitope mapping of polyclonal clotting factor VIII-inhibitory antibodies using phage display
Kessel et al. Humoral immune responsiveness to a defined epitope on factor VIII before and after B cell ablation with rituximab
Shima et al. Localization of the binding site for a factor VIII activity neutralizing antibody to amino acid residues Asp1663-Ser1669.
Di Giambattista et al. Mapping of natural anti-factor VIII antibodies in plasma pools from healthy donors: use of rationally designed synthetic peptides
Gharagozlou et al. Epitope specificity of anti-factor VIII antibodies from inhibitor positive acquired and congenital haemophilia A patients using synthetic peptides spanning A and C domains
AU2003260335B2 (en) Anti-idiotypic antibodies against factor VIII inhibitor and uses thereof
Di Giambattista et al. In silico prediction of FVIII epitopes recognised by natural autoantibodies in polyvalent immunoglobulin concentrates
Saenko et al. Molecular modifications in factor VIII concentrates produced from different plasma pools
Furlong et al. Monoclonal antibodies to factor VIII: their application in immunoblotting for the visualization of factor VIII in therapeutic concentrates and plasma
Gilles et al. Restricted epitope specificity of anti-FVIII antibodies that appeared during a recent outbreak of inhibitors
Meijer-Huizinga et al. Isolation and characterization of single-chain protein S
MÜHLE et al. and H. SCHNEIDER
Mühle et al. Characterization of Polyclonal Factor VIII-Inhibitory Antibodies

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20031030

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: CAF - DCF CVBA - SCRL

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: ISLER & PEDRAZZINI AG

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 60241094

Country of ref document: DE

Effective date: 20111124

REG Reference to a national code

Ref country code: NL

Ref legal event code: T3

REG Reference to a national code

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2372811

Country of ref document: ES

Kind code of ref document: T3

Effective date: 20120126

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110921

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110921

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110921

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111222

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120123

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110921

26N No opposition filed

Effective date: 20120622

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 60241094

Country of ref document: DE

Effective date: 20120622

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120531

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

Effective date: 20130131

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120531

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120506

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110921

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120506

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NL

Payment date: 20160519

Year of fee payment: 15

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: ES

Payment date: 20160512

Year of fee payment: 15

Ref country code: GB

Payment date: 20160520

Year of fee payment: 15

Ref country code: CH

Payment date: 20160519

Year of fee payment: 15

Ref country code: DE

Payment date: 20160520

Year of fee payment: 15

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: BE

Payment date: 20160519

Year of fee payment: 15

Ref country code: AT

Payment date: 20160520

Year of fee payment: 15

Ref country code: IT

Payment date: 20160524

Year of fee payment: 15

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 60241094

Country of ref document: DE

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: NL

Ref legal event code: MM

Effective date: 20170601

REG Reference to a national code

Ref country code: AT

Ref legal event code: MM01

Ref document number: 525470

Country of ref document: AT

Kind code of ref document: T

Effective date: 20170506

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20170506

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20170506

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20170531

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20170531

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20170601

REG Reference to a national code

Ref country code: BE

Ref legal event code: MM

Effective date: 20170531

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20170506

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20171201

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20170506

REG Reference to a national code

Ref country code: ES

Ref legal event code: FD2A

Effective date: 20180704

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20170507

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20170531